Transient changes in the intracellular calcium (Ca2+) concentration are known to be responsible for activating numerous physiological processes, including memory formation, muscle contraction, hormone secretion, fertilization, gene transcription and apoptosis. One striking phenomenon observed in numerous cell types, including cardiac myocytes, endothelial cells and eggs, is the generation of a series of regular calcium transients or oscillations in response to cellular stimuli. The best-studied example of this phenomenon is during mammalian fertilization, where calcium levels in the egg begin to oscillate in a regular fashion following fusion with the sperm.
These calcium oscillations occurring at fertilisation, sometimes referred to as the “calcium waves”, are believed to be the trigger for egg activation and consequent embryo development. Studies carried out over many years have attempted to discover and isolate the causative agent of this phenomenon with a view to using it for research and for a variety of practical applications, including diagnosis.
This striking Ca2+ signalling phenomenon in fertilized mammalian eggs arises from increases in inositol 1,4,5-trisphosphate (IP3) levels, which activates IP3 receptor-mediated Ca2+ release from intracellular stores in the egg. However, the basic mechanism involved in stimulation of phospho-inositide metabolism following sperm-egg interaction has not been determined in any species.
The ‘sperm factor hypothesis’ of signalling at fertilization proposes that spermatozoa contain a soluble Ca2+-releasing factor that enters the egg after the gamete membranes fuse together and generates Ca2+ oscillations. This is consistent with the finding that cytoplasmic fusion of sperm and egg is a prelude to Ca2+ release. Direct support for this hypothesis comes from experiments where micro-injection into eggs of either single spermatozoa or soluble sperm extracts triggers Ca2+ oscillations similar to those at fertilization in mammalian—and some non-mammalian—eggs. The mammalian sperm factor that generates Ca2+ oscillations is protein-based; acts across species; and can cause Ca2+ release in somatic cells as well as in cell-free systems, such as sea urchin egg homogenates. Sperm specifically express a Ca2+ oscillation-inducing protein, because micro-injecting messenger RNA (mRNA) isolated from spermatogenic cells, but not mRNA from other tissues, elicits fertilization-like Ca2+ oscillations in mouse eggs.
In intact eggs and egg homogenates, mammalian sperm extracts trigger Ca2+ release via stimulating IP3 production, indicating involvement of a phospho-inositide-specific phospholipase C (i.e. PI-PLC, usually referred to in short as PLC) in the signal transduction mechanism. The high level of PLC enzyme activity measured biochemically in sperm extracts has led some researchers to suggest that the sperm factor may itself be a PLC. However, the PLC-beta, gamma and delta (β, γ and δ) isoforms that exist in sperm are not detected in the chromatographic fractions of sperm extract that specifically cause Ca2+ oscillations. Also, when purified, recombinant PLCβ2, γ1 or δ1 proteins are added to egg homogenates, they fail to cause Ca2+ release. A PLCδ4 splice variant expressed in sperm has been shown to be involved in the acrosome reaction, rather than Ca2+ release in eggs at fertilization. Previous research in this field has been described in international patent specification no. WO 96/25495, to which reference should be made for a full understanding of, and as background to, the present application. The contents of WO 96/25495 are therefore incorporated herein by reference.
Patent specification no. WO 96/25945 assigned the cause of the above-mentioned calcium oscillations to a substance (a sperm factor) present in the equatorial segment of sperm, which was believed to diffuse into the egg after fusion therewith. This substance was identified as a 33 kD (approx.) protein of specified amino acid sequence. The nucleic acid coding for this protein was also specified. However, after cloning the gene and undertaking subsequent expression studies, it was concluded that this sperm factor candidate was unable to reconstitute calcium oscillations. A truncated form of the c-kit receptor, has also previously been a sperm factor candidate. However, neither these two, nor any other sperm proteins, have been shown to generate Ca2+ oscillations in eggs, the single-most distinctive feature of mammalian fertilization.
These observations have led some workers in the field to conclude, “sperm-derived PLC is not responsible for initiating Ca2+ release at fertilization” (Mehlmann et al in Dev Biol 236 492-501 (2001)), whilst others have stated: “the identification of this protein remains a problem for the next century of fertilization research” (Runft et al in Dev Biol 245 237-54 (2002)).